Characterization of the human cone transducin alpha-subunit gene and localization of itscis-acting transcriptional regulatory elements
Transducin is a member of a family of G-proteins which play pivotal roles in signal transduction. In photoreceptors, transducin relays the signal between the visual pigment proteins and the effector enzyme, cGMP-phosphodiesterase. Rod and cone photoreceptors each express their own transducin $\alpha$-subunits, GNAT1 and GNAT2, respectively. This work reports the characterization of the human GNAT2 gene as well as the localization of the cis-acting elements within its upstream region. The human GNAT2 transcription unit is 9967-base pairs (bp) in length and consists of eight exons with seven introns. The intron splice sites in the coding sequence for GNAT1 and GNAT2 are identical. Northern blot analysis of RNA from human retinas and a retinoblastoma cell line, WERI-Rb1, reveals a 1.7-kb transcript for GNAT2. Multiple transcription initiation sites, spanning 31-bp, were mapped for human retina and WERI-Rb1 RNA. The promoter region contains several common consensus sequences, including a TATA box sequence at $-$29. The GNAT2 upstream sequence shows no significant identity with the upstream region of the human GNAT 1 gene or with the upstream regions of the color visual pigment genes indicating that the expression of GNAT2 may be regulated differently than these other rod- and cone-specific proteins. Transfection of nested deletions of the GNAT2 upstream region cloned 5$\sp\prime$ of the chloramphenicol acetyltransferase gene into WERI-Rb1 cells demonstrated the presence of a strong silencer between $-$1130 and $-$23 and a weak promoter activity between $-$151 and $-$10. The promoter showed cell-specificity by not expressing in HeLa cells while the silencer was just as active. Three cis-acting elements were found in the silencer using DNaseI footprinting. Mobility-shift assays using nuclear proteins from either WERI-Rb1 or HeLa cells generally gave the same size of protein-DNA complexes for each footprinted sequence suggesting that these sequences bind the same proteins in these cells. Methylation interference for G residues in consensus sequences within these footprints also suggests that some of these silencer elements are binding common transcription factors.
Bridges, Purdue University.
Off-Campus Purdue Users:
To access this dissertation, please log in to our